The ability to detect and identify trace quantities of chemicals has become increasingly important in virtually every scientific discipline, ranging from part per billion analyses of pollutants in sub-surface water to analysis of cancer treatment drugs in blood serum. Surface-enhanced Raman spectroscopy (SERS) has proven to be one of the most sensitive methods for performing such chemical analyses by the detection of a single molecule (see Nie, S. and S. R. Emory, xe2x80x9cProbing Single Molecules and Single Nanoparticles by Surface Enhanced Raman Scatteringxe2x80x9d, Science, 275,1102 (1997)). A Raman spectrum, similar to an infrared spectrum, consists of a wavelength distribution of bands corresponding to molecular vibrations specific to the sample being analyzed (the analyte). For example, appended FIG. 3 (to be discussed more fully below) shows the infrared and Raman spectra of adenine. In the practice of Raman spectroscopy, the beam from a light source, generally a laser, is focused upon the sample to thereby generate inelastically scattered radiation, which is optically collected and directed into a wavelength-dispersive spectrometer in which a detector converts the energy of impinging photons to electrical signal intensity.
Historically, the very low conversion of incident radiation to inelastic scattered radiation limited Raman spectroscopy to applications that were difficult to perform by infrared spectroscopy, such as the analysis of aqueous solutions. It was discovered in 1974 however that when a molecule in close proximity to a roughened silver electrode is subjected to a Raman excitation source the intensity of the signal generated is increased by as much as six orders of magnitude. (see Fleischmann, M., Hendra, P. J., and McQuillan, A. J., xe2x80x9cRaman Spectra of Pyridine Adsorbed at a Silver Electrode,xe2x80x9d Chem. Phys. Lett, 26, 123, (1974), and Weaver, M. J., Farquharson, S., Tadayyoni, M. A., xe2x80x9cSurface-enhancement factors for Raman scattering at silver electrodes. Role of adsorbate-surface interactions and electrode structure,xe2x80x9d J. Chem. Phys., 82, 4867-4874 (1985)). The mechanism responsible for this large increase in scattering efficiency has been the subject of considerable research (see Section Four: Theory in xe2x80x9cSurface-Enhanced Raman Scattering,xe2x80x9d [M. Kerker and B. Thompson Eds.] SPIE, MS 10, also p. 225 (1990)). A description of the theory is given by B. Pettinger in xe2x80x9cLight Scattering by Adsorbates at Ag Particles; Quantum-Mechanical Approach for Energy Transfer Induced Interfacial Optical Processes Involving Surface Plasmons, Multipoles, and Electron-hole Pairs,xe2x80x9d J. Chem. Phys., 85, 7442-7451 (1986). Briefly, incident laser photons couple to free conducting electrons within the metal which, confined by the particle surface, collectively cause the electron cloud to resonate. The resulting surface plasmon field provides an efficient pathway for the transfer of energy to the molecular vibrational modes of a molecule within the field, and thus generates Raman photons (see xe2x80x9cSurface-Enhanced Raman Scattering; Section Four: Theoryxe2x80x9d, supra).
The described phenomenon occurs however only if the following three conditions are satisfied: (1) that the free-electron absorption of the metal can be excited by light of wavelength between 250 and 2500 nanometers (nm), preferably in the form of laser beams; (2) that the metal employed is of the appropriate size (normally 5 to 1000 nm diameter particles, or a surface of equivalent morphology), and has optical properties necessary for generating a surface plasmon field; and (3) that the analyte molecule has effectively matching optical properties (absorption) for coupling to the plasmon field (see Weaver, J. Chem. Phys., 82, 4867-4874 (1985), and Pettinger, J. Chem. Phys., 85, 7442-7451 (1986), supra). Although limited signal enhancement has been observed for the other coinage metals, such as nickel and platinum, as well as for alloys containing one or more of the coinage metals, as a practical matter the foregoing conditions restrict SERS to the Periodic Table Group IB metals, copper, gold, and silver, with diameters between 5 and 200 nm (see Pettinger, J. Chem. Phys, 85, 7442-7451 (1986) supra, and Wang, D. -S., and Kerker, M., xe2x80x9cEnhanced Raman Scattering by Molecules Adsorbed at the Surface of Colloidal Spheroids,xe2x80x9d Physical Review B., 24, 1777-1790 (1981)). The SERS method has been used to measure the spectra of adenine on a silver-doped sol-gel coated glass substrate, and has achieved signal increases of six orders of magnitude, as shown by appended FIG. 3(c).
Analyses for numerous chemicals and biochemical by SERS has been demonstrated using: (1) activated electrodes in electrolytic cells (see Lombardi, D. R., C. Wang, B. Sun, A. W. Fountain III, T. J. Vickers, C. K. Mann, F. R. Reich, J. G. Douglas, B. A. Crawford, and F. L. Kohlasch, Appl. Spectrosc. 48, 875-833 (1994); Storey, J. M. E., Shelton, R. D., Barber, T. E., and Wachter, E. A., xe2x80x9cElectrochemical SERS Detection of Chlorinated Hydrocarbons in Aqueous Solutions,xe2x80x9d Appl. Spectrosc., 48, 1265-1271 (1994); Freeman, R. D., Hammaker, R. M., Meloan, C. E., and Fately, W. G., xe2x80x9cA detector for liquid chromatography and flow injection analysis using SERS,xe2x80x9d Appl. Spectrosc., 42, 456-460 (1988); Angel, S. M., L. F. Katz, D. D. Archibold, L. T. Lin, D. E. Honigs, App. Spectrosc. 42, 1327 (1988); and Vo-Dinh, T., Stokes, D. L., Li, Y. S., and Miller, G. H., xe2x80x9cFiber-Optic Sensor Probe For In-Situ Surface-Enhanced Raman Monitoring,xe2x80x9d SPIE, 1368, 203-209 (1990)); (2) activated silver and gold colloid reagents (see Berthod, A., J. J. Laserna, and J. D. Winefordner, xe2x80x9cSERS on silver hydrosols studied by flow injection analysisxe2x80x9d, Appl. Spectrosc. 41, 1137-1141 (1987) 42, 456-460 (1988) and Angel, S. M., L. F. Katz, D. D. Archibold, L. T. Lin, D. E. Honigs, Appl. Spectrosc. 42, 1327 (1988); and (3) activated silver and gold substrates (see Vo-Dinh, SPIE, 1368, 203-209 (1990), and Storey, J. M. E. Barber, T. E., Shelton, R. D., Wacher, E. A., Carron, K. T., and Jiang, Y. xe2x80x9cApplications of Surface-Enhanced Raman Scattering (SERS) to Chemical Detectionxe2x80x9d, Spectroscopy, 10(3), 20-25 (1995). None of the foregoing techniques is capable of providing quantitative measurements, however, and consequently SERS has not gained widespread use.
More specifically, the first technique referred to uses electrodes that are xe2x80x9croughenedxe2x80x9d by changing the applied potential between oxidation and reduction states; it is found that the desired metal surface features (roughness) cannot be reproduced faithfully from one procedure to the next, and the method is also limited to electrolyte solutions. In the second technique, colloids are prepared by reducing a metal salt solution to produce metal particles, which in turn form aggregates. Particle size and aggregate size are strongly influenced by initial chemical concentrations, temperature, pH, and rate of mixing, and again therefore the desired features are not reproducible; also, the method is limited to aqueous solutions. Finally, the third technique mentioned uses substrates that are prepared by depositing the desired metal onto a surface having the appropriate roughness characteristics. To permit the analysis, the sample is preferably dried on the surface to concentrate the analyte on the active metal, and once again replication is difficult to achieve; the colloids and substrates are further limited moreover in that the chemical interaction of the analyte and the SER-active metal is not reversible, thus precluding use of the materials for repeat measurements. The relative merits of the three methods described above, for preparing SER-active surfaces, have been further reviewed by K. L. Norrod, L. M. Sudnik, D. Rousell, and K. L. Rowlen in xe2x80x9cQuantitative comparison of five SERS substrates: Sensitivity and detection limit,xe2x80x9d Appl. Spectrosc., 51, 994-1001 (1997).
It is therefore the broad object of the present invention to provide a novel method for preparing a SERS-active material that, in general, avoids the deficiencies of the techniques heretofore known and that enables reproducible, reversible, and quantitative measurements to be performed with a high level of accuracy.
A more specific object of the invention is to provide a novel method for incorporating metal particles within a sol-gel matrix so as to produce a SERS-active material having the foregoing features and advantages.
Other more specific objects of the invention are to provide a novel SERS-active material that is not restricted to specific environments, such as electrolytes, particular solvents, or as evaporates on surfaces, and to provide a method for the preparation thereof.
A further object of the invention is to provide novel sample holders (e.g. internally coated vials and multiple-well micro-sample plates) sampling systems (e.g., internally coated tubing for sample flow), and sample probes (e.g., externally coated rods or fiber optics that can be placed into a sample), suitable for use in performing SERS analyses.
Yet another object of the invention is to provide a novel chemical analysis technique using surface-enhanced Raman spectroscopy and, in particular, such an analysis technique that employs the sample holders, systems, and probes fabricated in accordance herewith.
The foregoing and related objects of the invention are achieved, in general, by providing a chemical synthesis route for incorporating a metal within a sol-gel, such that interactions between impinging radiation and analyte molecules at the surface of the metal within the prepared sol-gel are enhanced, so as to in turn enhance the efficiency with which inelastically scattered (Raman) photons are generated. The chemical route described provides a means for optimally selecting the metal particle, the size of the particles, and the concentration of the metal within the sol-gel; it provides means for effectively controlling the porosity (pore-size and number of pores) and polarity (charge) of the sol-gel; and it enables coating of surfaces of virtually any shape, during gelation, such that a variety of sample holders, systems, and probes can be fabricated.
More particularly, certain objects of the invention are achieved by the provision of a method for producing a metal-doped sol-gel material, effective for surface-enhanced Raman spectroscopy, comprising the steps:
(a) providing a liquid formulation reactive for gelation to produce a sol-gel, the formulation comprising water; a matrix-forming component including a compound having the chemical formula M(OR)n, wherein M is a metal selected from the group consisting of silicon, aluminum, titanium, zirconium, and mixtures thereof, R is an organic group, and n is of course an integer; ions of a SERS-active metal; and, optionally, a cosolvent for the water and the matrix-forming component;
(b) effecting gelation and drying of said formulation, at a temperature not in excess of 100xc2x0 C., to produce a porous sol-gel material; and
(c) treating at least one surface of said sol-gel material to effect reduction of said metal ions present at said surface to elemental-state particles.
It will be appreciated that gelation and drying (to remove water, with or without any residual organic solvent) may be effected as a continuous operation or as separate gelation and drying steps.
The matrix-forming component will preferably comprise a metal alkoxide, the organic group of which is a short alkyl chain, and the cosolvent employed will usually be a short chain (lower) alcohol. Preferably, the matrix-forming component will comprise tetramethylorthosilicate (TMOS), tetraethylorthosilicate (TEOS), or methyltrimethoxysilane (MTMS), and may desirably comprise a mixture thereof. In most instances the mole ratio of water to matrix-forming component will be in the range 0.01-50:1, and the mole ratio of cosolvent (when used) to water will be in the range 10-0.01:1.
The SERS-active metal ion will normally constitute about 0.1 to 15 mole percent of the formulation, and generally it will be of a Periodic Table Group IB metal. The metal ion will advantageously be introduced into the formulation as an aqueous solution of a complex, such as a silver amine complex. The elemental-state particles should have a diameter in the range 5 to 1000 nanometers and, when the ion is of a readily oxidizable metal, the step of gelation and drying will most desirably be carried out in an oxygen-starved atmosphere.
Other objects of the invention are attained by the provision of a method that includes the additional step of applying the liquid formulation to a substrate. Upon gelation, drying and reduction of the metal ion, the treated sol-gel material and substrate comprise a sensor for receiving, on at least the xe2x80x9conexe2x80x9d surface, an analyte for Raman spectral analysis.
Additional objects are attained by the provision of a sensor produced by the method of the invention. Suitable substrates include slides, vials, multi-well micro-sample plates, tubes, optical elements (such as fiber optics, lenses, mirrors, and the like), and probe elements.
Further objects of the invention are attained by the provision of a method for carrying out surface-enhanced Raman spectral analysis of an analyte, comprising: providing the sol-gel material described; depositing an analyte upon the xe2x80x9conexe2x80x9d surface of the sol-gel material; illuminating the one surface with radiation of at least one wavelength that is effective for causing the elemental-state particles to produce a plasmon field and for causing the field to interact with molecules of the analyte, to produce Raman photons; and collecting inelastically scattered radiation emitted from the one surface, for spectral analysis.
Still further objects are attained by the provision of Raman instrument comprising: a radiation source; a detector for detecting inelastically scattered radiation; a surface-enhanced Raman sensor produced in accordance herewith; and optics for directing radiation from the source upon the sensor, and for directing radiation emitted from the sensor upon the detector. The radiation source employed will usually produce radiation in the wavelength range of 250 to 2500 nanometers, and the elemental-state particle will have a diameter in the range 5 to 1000 nanometers; preferably, the particles will be of a Group IB metal.